Obtaining high-expression high-heat-stability enzymes based on the important influence of expression amount and heat stability on the application performance of enzymes has been the research hotspot in the field of enzyme engineering.
There is a certain mutual promotion relationship among expression level, heat stability and enzyme catalytic activity. In terms of expression level, the prior art generally increases the expression level of protease by the optimization of the expression element or the fusion of expression tags at the terminal, but the optimization of the expression element has certain uncertainty, and the most commonly used expression tags, such as MBP, GST and other macromolecules, all have certain effects on the late application of the enzyme, and often require complex procedures to be removed. In terms of stability, with the development of structural biology and bioinformatics, researchers can accurately locate the amino acid residues or peptides that affect the heat stability of the enzyme molecules by analysis of some structural parameters (such as B-factor, RMSF value, etc.) or comparison of homologous sequences, so that site-directed mutagenesis is carried out to enhance the heat stability of enzymes. Although it has become a conventional strategy for the heat stability modification of enzymes, the above molecular modification technology still has inherent technical defects. The premise of site-directed mutagenesis is to obtain accurate information on the molecular structure of the enzyme, so in vitro directed evolution is faced with a large number of mutant screenings, resulting in the difficulty in obtaining mutants with significantly improved heat stability in a short period of time.
Therefore, the establishment of an efficient and convenient enzyme stabilization strategy has become the focus of researchers at home and abroad.
It is worth noting that the increase in the expression level and heat stability of the enzyme by the fusion short peptide is accomplished under the conditions of no enzyme structure information and a large number of mutant screenings, and the efficiency is significantly improved as compared with the traditional molecular modification technology. It was first discovered by the Urabe team at Osaka University in Japan when studying the heat stability of Bacillus stearothermophilus catalase (Nat Biotechnol, 1999, 17(1): 58-61); the researchers randomly fused short peptides of different lengths and amino acid sequences at the C-terminal of catalase to obtain a series of high-heat-stability mutants; and then, the Kanaya team at Osaka University discovered that the fusion of the Pyrococcus horikoshii ribonuclease C-terminal heptapeptide (IGCIILT) at the C-terminal can improve the heat stability of ribonuclease from different sources to different degrees (PLoS ONE, 2011, 6(1): e16226).
SAPs are a category of short peptides with alternately distributed hydrophilic and hydrophobic amino acids that can be spontaneously assembled into nanostructures. Due to unique amphipathic properties, the short peptides can form hydrogels in water, thereby immobilizing the target proteins or other small molecules. Based on this, in the preliminary study of this laboratory (Appl Microbiol Biot, 2013, 97(21): 9419-9427), a category of SAPs were fused to the N-terminal of the enzyme for heterologous expression of the fusion enzyme, and it was found that SAPs have the function of increasing the expression level and stability of enzymes, and S1 (AEAEAKAKAEAEAKAK) with special charge distribution has certain universal effects; Lin et al. (Faraday Discuss, 2013,166:233) of Tsinghua University used SAP (LELELKLKLELELKLK), which is similar to the amino acid composition of S1, to fuse at the end of the enzyme, which can promote the formation of active inclusion bodies, indicating that the amino acid composition has an important influence on the secretion expression and stability of SAPs fusion protein. On the other hand, the composition of the linker between SAPs and the enzyme also plays an important role in the expression and stability of the fusion enzyme (Enzyme Microb Tech, 2016,82:105-109).
The present disclosure attempts to construct a polypeptide library based on SAPs to quickly, efficiently and conveniently obtain an enzyme mutant having high expression, high activity and high stability.